Novel N-benzylpiperidine carboxamide derivatives as potential cholinesterase inhibitors for the treatment of Alzheimer's disease

European Journal of Medicinal Chemistry 179 (2019) 680e693

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Novel N-benzylpiperidine carboxamide derivatives as potential cholinesterase inhibitors for the treatment of Alzheimer's disease Divan G. van Greunen a, C. Johan van der Westhuizen a, Werner Cordier b, Margo Nell b, Andre Stander c, Vanessa Steenkamp b, Jenny-Lee Panayides d, **, Darren L. Riley a, * a

Department of Chemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, Lynnwood Road, Pretoria, South Africa Department of Pharmacology, Faculty of Health Sciences, University of Pretoria, Bophelo Road, Pretoria, South Africa Department of Physiology, Faculty of Health Sciences, University of Pretoria, Lynnwood Road, Pretoria, South Africa d Pioneering Health Sciences, CSIR Biosciences, Meiring Naud
e Road, Pretoria, South Africa b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 May 2019 Received in revised form 28 June 2019 Accepted 28 June 2019 Available online 1 July 2019

A series of fifteen acetylcholinesterase inhibitors were designed and synthesised based upon the previously identified lead compound 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl 1-benzylpiperidine4-carboxylate (5) which showed good inhibitory activity (IC50 0.03 ± 0.07 mM) against acetylcholinesterase. A series of compounds were prepared wherein the ester linker in the original lead compound was exchanged for a more metabolically stable amide linker and the indanone moiety was exchanged for a range of aryl and aromatic heterocycles. The two most active analogues 1-benzyl-N-(5,6-dimethoxy-8Hindeno[1,2-d]thiazol-2-yl)piperidine-4-carboxamide (28) and 1-benzyl-N-(1-methyl-3-oxo-2-phenyl2,3-dihydro-1H-pyrazol-4-yl) piperidine-4-carboxamide (20) afforded in vitro IC50 values of 0.41 ± 1.25 and 5.94 ± 1.08 mM, respectively. In silico screening predicts that 20 will be a blood brain-barrier permeant, and molecular dynamic simulations are indicative of a close correlation between the binding of 20 and the Food and Drug Administration-approved cholinesterase inhibitor donepezil (1). © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Acetylcholinesterase Alzheimer's disease Benzylpiperidine Butyrylcholinesterase Carboxamide Cytotoxicity Donepezil

1. Introduction Alzheimer's disease (AD) is the most common form of dementia accounting for an estimated 60e80% of all cases in the United States of America (USA) [1]. In 2017, it was estimated that 5.3 million Americans aged 65 and older were living with AD, which is predicted to increase to 13.8 million by 2050 [1]. Alzheimer's disease is neurodegenerative in nature and characterised by a gradual onset and progression of deficits in more than one area of cognition, including disruption in behavioural, language and memory skills [2,3]. To date, consensus on the cause of AD has not been reached, however, several hypotheses have been put forward based upon the various pathophysiological factors observed. One of the most widely investigated being the cholinergic hypothesis [3,4], which is linked to the observation that AD is

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (J.-L. Panayides), [email protected] (D.L. Riley). https://doi.org/10.1016/j.ejmech.2019.06.088 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

characterised by a deficiency of the neurotransmitter acetylcholine. The deficiency arises either as a result of a decreased production of acetylcholine, or through the amplification of the enzymatic degradation thereof by acetylcholinesterase (AChE), and to a lesser extent, butyrylcholinesterase (BuChE) [5,6]. Furthermore, AChE has also been shown to bind to amyloid-ß (Ab) and play a role in the deposition of Ab plaques, another hallmark of AD [7]. As a result, the inhibition of AChE is regarded as an important neuroprotective target in AD drug discovery, acting by increasing relative acetylcholine concentrations in the brain and reducing the deposition of toxic Ab plaques [7]. Currently, three AChE inhibitors, donepezil 1, rivastigmine 2 and galantamine 3 (Fig. 1), are approved by the US Food and Drug Administration (FDA) for the treatment of the symptoms associated with mild to advanced AD [8], with a fourth, tacrine 4, having previously been discontinued due to concerns linked to hepatotoxicity [9]. The AChE enzyme consists of two domains, the active catalytic anionic site (CAS) at the bottom of the binding pocket and a peripheral anionic site (PAS) at the entrance to the pocket [10]. During the development of donepezil 1, Sugimoto and co-workers focused

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donepezil 1 are described, and in vitro inhibition against AChE and BuChE as well as cytotoxicity are reported. In addition, in silico assessment of compound-binding using molecular docking and molecular dynamic (MD) simulations, is described. 2. Chemistry

Fig. 1. Current and former FDA approved AChE inhibitors for the treatment of AD [8].

largely on the optimisation of binding in the CAS, showing that an N-benzylpiperidine moiety was critical for activity affording strong binding interactions with the Trp84, Trp279, Phe330 and Phe331 residues in the active site. In addition, the dimethoxy indanone moiety has indicated binding interactions with Phe288 and Trp279 in the PAS site [10]. Subsequent structure activity relationship (SAR) studies have been undertaken involving the exploration of chemical space surrounding donepezil 1. In several examples, the indanone moiety has been replaced with different heterocycles including coumarin, isoindoline-1,3-dione and phthalimide [11e14] and in some cases have shown that concomitant inhibition of Ab aggregation is linked to systems displaying binding to the PAS site [12,13]. In previous work reported by our group, the N-benzylpiperidine ring system of donepezil 1 was replaced with a range of different saturated N-benzylated ring systems and the methyl bridge between the indanone and the N-benzylpiperidine ring system with various different linkers (Fig. 2) [15]. The SAR conducted identified compound 5 as the most active ligand with a half-maximal inhibitory concentration (IC50) of 0.03 ± 0.07 mM with no observable cytotoxicity in the SH-SY5Y neuroblastoma cell line (IC50 of >100 mM). In the present study, compound 5 was modified by replacing the ester linkage (Part B) with that of an amide linker to reduce the metabolic liability associated with the ester. Furthermore, to explore the effect of binding at the PAS site, the indanone moiety (Part A) was replaced with several aromatic and aryl heterocycles. In this study, the synthesis of fifteen novel analogues of

The preparation of the desired carboxamide derivatives was envisaged using standard amide coupling chemistry to allow the coupling of imidazole 6 with various commercial and synthetic heterocyclic amines. The N-benzylpiperidine imidazole derivative 6 was prepared in three steps from ethyl isonipecotate 7 [16,17], initial treatment with benzyl chloride afforded the N-benzylated ester 8, thereafter, hydrolysis in the presence of methanolic sodium hydroxide afforded acid 9 (Scheme 1). Subsequent treatment with carbonyl diimidazole afforded 6 in a 71% yield over three steps. Initially, it was envisaged to use this approach to access the amide analogue 10 of previously identified compound 5. To access the required amine 11, 5,6-dimethoxy-1-indanone 12 was treated with isopentyl nitrite in an acidic methanolic solution affording the oximino compound 13 as a precipitate in 81% yield (Scheme 2) [18]. Thereafter, catalytic reduction under acidic conditions afforded the amine as an ammonium salt 11 in 95% yield [19]. Unfortunately, subsequent coupling attempts proved unsuccessful. Interestingly, the difficulties noted with the preparation of 10 did not appear to be general, and a range of carboxamide derivatives 14e28 were readily prepared by coupling 6 with commercial and synthetic amines under mild conditions in the presence of catalytic 4-(dimethylamino)pyridine (4-DMAP) (Scheme 3, Table 1) in low to good yields [20]. In the case of carboxamide 28, the required substituted aminothiazole heterocycle 29 was prepared by bromination of 5,6dimethoxy-1-indanone 12 [15,21], affording the a-halogenated indanone 30 followed by treatment with thiourea to afford 29 in a 70% yield across two steps (Scheme 4). Thereafter, the coupling required the initial deprotonation of amine 29 with sodium hydride prior to reaction with 6 affording carboxamide 28 in 12% yield. 3. Structure activity relationship study The inhibitory activity of all compounds was assessed against Electrophorus electricus AChE (EeAChE) and BuChE from equine serum (eqBuChE) using Ellman's spectrophotometric method [22] with some minor modifications [23], galantamine was used as the positive control. The anticholinesterase activities for the synthesised compounds expressed as IC50 values are provided in Table 1. In addition, all compounds were assessed for cytotoxicity in the SH-SY5Y neuroblastoma cell line using the sulforhodamine B (SRB) staining assay as described by Vichai and Kirtikara [24],

Fig. 2. Progression from previously reported work involving the incorporation of an amide linker and the replacement of the indanone moiety with different heterocyclic systems [15].

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Scheme 1. (i) 1.05 eq. BnCl, 3 eq. Et3N, CH3CN, reflux, 3 h, 88%, (ii) 2 eq. NaOH, MeOH, reflux, 24 h, 98%, (iii) 1.2 eq. carbonyl diimidazole, CH2Cl2, 0  C, 2 h, 82%.

Scheme 2. (i) 1 eq. isopentyl nitrite, conc. HCl, MeOH, 40  C, 30 min, 81%, (ii) H2, Pd/C, 1.5 eq. conc. HCl, EtOH, rt, 1 h, 95%, (iii) 6 eq. NaH, DMSO, rt, 3 h, 0%.

4. In silico screening 4.1. Molecular docking

Scheme 3. (i) 1 eq. ReNH2, 0.6 eq. 4-DMAP, 1 eq. Et3N, CH2Cl2, rt e reflux, 12 h.

although the SH-SY5Y cell line is cancerous in nature, it does present as a model of a neurological cellular environment [24]. Two compounds were found to have EeAChE IC50 values of less than 10 mM, namely 20 (IC50 ¼ 5.94 mM) and 28 (IC50 ¼ 0.41 mM), with the latter having 5-fold less activity than donepezil. In both instances, 20 and 28 have a proton acceptor in a similar position to the indanone carbonyl in 1 and 5, suggesting, that although the PAS is not the primary binding site, it is still crucial for the development of efficient AChE inhibitors. It was further noted, that the position of the h-bond acceptor binding to the PAS site appears to play a role in inhibition. When the nitrogen was changed from an ortho position to that of meta or para position in 21e23, an approximate 50% decrease in activity was noted. Interestingly, thiazole 18 exhibited an IC50 of 51.10 mM, however, when converted to the analogous tricyclic 28 there was a 125-fold increase in activity to 0.41 mM. This trend suggests that binding in the PAS is highly susceptible to subtle changes in the electronic nature of the proton acceptor and/or that the additional aromatic ring systems are improving binding, through p-p stacking. In all cases, activity was selective for AChE with no observable inhibition of BuChE below 100 mM, furthermore, compounds 20 and 28 did not display appreciable cytotoxicity with IC50 values of 31.31 ± 1.32 mM (selectivity index of 76) for 28 and > 100 mM for 20.

Preliminary estimation of the binding affinity of the compounds was calculated using the binding energy according to the combined Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method. Molecular docking was performed using the crystal structure of Torpedo eel AChE (PDB: 1EVE) [25] using Glide XP and €dinger program suite [26,27]. The Prime MM-GBSA from the Schro binding pocket comprises the CAS containing Trp84 and Phe330, while the PAS includes the Tyr70, Asp72, Try 121, Tyr334 and Trp279 residues. The crystal structure contains donepezil 1 bound into the 20 Å deep gorge which is highly solvated, with the R-configuration of the alpha carbon of the indanone carbonyl being noted. Donepezil 1 was docked back into the crystal structure and it was noted that several of the water molecules needed to be included during the docking process to obtain the correct binding pose (RMSD of redocked pose: 0.918 Å). This resulted in water-bridged interactions between the ligand and the residues within the binding pocket. Thus, the water bridge network was kept as all the synthesised ligands have an N-benzylpiperidine moiety in common with donepezil 1 around which several water molecules are located. The common scaffold, N-benzylpiperidine found in donepezil 1 and the synthesised compounds produced similar poses and interactions, with the exception of compounds 16, 21 and 27, which formed binding poses in the reverse orientation. These poses had N-benzylpiperidine interactions with the PAS residues and the analogue structures deep within the binding pocket at the CAS. When all the structural waters within the binding pocket were included, docking and MM-GBSA calculations provided similar energies for the ligands entering the pocket from different orientations. Thus, poses were visually inspected to select the correct

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Table 1 In vitro AChE, BuChE inhibitory activity and cytotoxicity of compounds 14e28..

Yield (%)

EeAChE IC50 ± SEM (mM)

eqBuChE IC50 ± SEM (mM)

Cytotoxicity IC50 ± SEM (mM)

14

91

25.60 ± 1.10

>100

40.48 ± 1.21

15

90

13.98 ± 1.16

>100

13.57 ± 1.15

16

85

>100

>100

73.42 ± 1.35

17

99

44.39 ± 1.17

>100

50.90 ± 1.58

18

59

51.10 ± 1.12

>100

76.62 ± 1.11

19

93

>100

>100

>100

20

34

5.94 ± 1.08

>100

>100

21

77

30.88 ± 1.19

>100

>100

22

59

58.89 ± 1.15

>100

>100

23

77

58.18. ± 1.14

>100

3.37 ± 1.19

24

97

>100

>100

>100

25

53

>100

>100

99.32 ± 1.32

26

38

>100

>100

>100

27

59

82.10 ± 1.14

>100

>100

Compound

R

(continued on next page)

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Table 1 (continued ) Yield (%)

EeAChE IC50 ± SEM (mM)

eqBuChE IC50 ± SEM (mM)

Cytotoxicity IC50 ± SEM (mM)

28

12

0.41 ± 1.25

>100

31.31 ± 1.32

Donepezil 1 Galantamine 3

e e

0.08 ± 0.01 3.58 ± 1.24

5.20 ± 0.01 e

95.13 ± 1.30 e

Compound

R

Scheme 4. (i) 1.05 eq. Br2, MeOH, rt, 30 min, 84%, (ii) 1 eq. thiourea, 5 eq. 3 M aq. NH3, H2O, rt-reflux, 12 h, 83%.

conformation. The synthesised ligands with the correct binding pose had comparable binding energy when compared with that of donepezil 1. For example, the binding energy of 28 was 66.4 kcal/ mol whereas donepezil 1 was 63.4 kcal/mol. An overlay of the best pose for donepezil 1, 20 and 28 (which displayed the highest inhibitory activity against AChE) shows that the N-benzylpiperidine moiety, which is common to all three, overlaps almost perfectly (Figs. 3 and 4) with analogous p-p stacking interactions between the benzyl ring and Trp84. The charged nitrogen on the piperidine interacts with Trp84, Tyr334 and Phe330 via p-cation interactions and hydrogen bonding through a water bridge is noted for the protonated nitrogen to Try121. While donepezil 1 forms a second water bridge to Phe288 and Phe331, 28 has a second hydrogen bond to Tyr121. The primary amide of 28 lies perpendicular between the two cyclic structures (piperidine and tricyclic indenothiazole ring systems), which

Fig. 4. Overlaid docked pose of donepezil 1 (green) and compound 20 (white) in torpedo eel AChE. Both compounds have p-p stacking interactions with Trp84 shown in blue, while donepezil has an additional stacking interaction with Trp279. p-Cation interactions with Try334, Phe330 and Trp84 for both complexes are shown in green. Furthermore, both compounds share water bridged interactions with HOH1254 to Phe288 and Phe331. Only selected residues and water molecules are shown within the binding pocket for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Overlaid docked poses of donepezil 1 (green) and compound 28 (white) in torpedo eel AChE. In both instances' compounds have p-p stacking interactions with Trp279 and Trp84 shown in blue, and the p-cation interactions with Tyr334, Phe330 and Trp84 shown in green. HOH1159 acts as a water bridge between Tyr121 and the protonated nitrogen on the piperidine ring of both compounds shown in yellow. Compound 1 forms an additional water bridge with HOH1254 to Phe288 and Phe331, while 28 has a hydrogen bond between the secondary amine and Trp121 as well as has p-p stacking between the heterocycle and Tyr121. Only selected residues and water molecules are shown within the binding pocket for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

allows the nitrogen to form a hydrogen bond with the hydroxyl on Tyr121 at 2.18 Å. Further p-p stacking is noted between compounds 1 and 28 with the PAS residue Trp279. In the case of 28 a second pp stacking interaction with Tyr121 is observed which is not possible with ligand 1, as the cyclopentanone substructure within indanone ring of 1 is not aromatic, suggesting that the aforementioned differences between compounds 18 and 28 (Table 1) are arising due to additional p-p stacking interactions present in 28. In contrast, compound 20 and donepezil 1 share mostly similar interactions with Trp84, Tyr334, Phe330, Trp84, Phe288, Phe331 and Trp279 (Figs. 3 and 4). 4.2. Molecular dynamics simulations In order to assess how ligand-enzyme interactions vary over time, 40 ns molecular dynamic simulations were performed at 310 K for AChE complexed with 1, 28 and 20. Analysis of the interactions between the compound and the protein during the simulation were done using the simulation interaction diagrams

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€dinger, as depicted in Fig. 5(aec), for 1, 28 and 20 tool of Schro respectively (refer to Fig. 6 for a schematic representation of the various interactions). Compound 28 is shown to have an increased frequency of interactions with various residues (Try70, Asp72, Tyr 121, Ser286, Phe330, Phe331 and Tyr334) but a decrease in interaction frequency was noted with Trp279 and Phe288 when compared to that of donepezil 1. This loss in interaction frequency for 28 is ascribed to the tricyclic indenothiazole ring system which spins/rotates during the simulation and does not show any preference to stay in a fixed planar orientation. Interestingly, compound 20 is shown to have increased frequency of interactions with Tyr121, Phe288, Arg289, Phe330, Phe331 and Tyr334 when

685

compared with donepezil 1, and unlike 28 there are no major losses in interactions. The variation of the dihedral angle between the PAS binding heterocycle (Part A, Fig. 2) and the CAS binding benzyl piperidine group of 1, 28 and 20 (Fig. 7) affords a more in-depth understanding of the ligand-enzyme interactions. It is apparent that 1 prefers a torsional angle of approximately 90 due to the water bridged assisted hydrogen bond between the ketone of the indanone and Phe288, preventing the indanone from rotating during the simulation. A similar affect is noted for 20 where the torsional angle is also kept at approximately 90 due to the water bridges between the cyclic amide carbonyl and Arg289 and Phe288. In contrast, in

Fig. 5. Protein-ligand contacts monitored during a 40 ns MD simulation at 310 K for the (a) AChE-1 complex, (b) AChE-28 complex and (c) AChE-20 complex. The contacts are categorised into four types: hydrogen bonds (green), hydrophobic (purple), ionic (red) and water bridges (blue), with the frequency of the contacts which the ligand makes residues in the binding pocket shown. The stacked bar charts of the contacts are normalised, with values over 1.0 representing protein residues that make multiple contacts with the ligand. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 6. Schematic representation showing interactions which the ligands (a) donepezil 1, (b) 28 and (c) 20 make with the surrounding residues of AChE during a 40 ns MD simulation at 310 K. Interactions that occur in more than 14% of the simulation time are shown.

the case of 28, the carbonyl present is located closer to the piperidine ring than in the case of 1, preventing interactions with Phe288, instead forming infrequent interactions with Tyr70 and Asp72. Furthermore, although the nitrogen and sulphur atoms within the tricyclic ring system can act as h-bond acceptors they do not show a clear interaction preference, resulting in the indenothiazole tricyclic system rotating by ~310 during the simulation for 28. 4.3. Pharmacokinetics, ADME parameters and drug-like nature The library of compounds was further assessed in terms of physiochemical descriptors, predictive absorption, distribution, metabolism and excretion (ADME) parameters and pharmacokinetic properties using the Swiss Institute of Bioinformatics SwissADME web tool [28]. All compounds prepared showed good druglikeness in terms of the Lipinski guidelines and no pan assay interference structures (PAINS) were noted [29]. Ligands 14e27

were computed to have total polar surface areas (TSPA) in the range of 32e73 Å and a WLogP in the range of 0.43e3.56, suggesting that they will act as blood brain barrier (BBB) permeants (Fig. 8). In the case of the most active ligand identified 28 (IC50 0.41 mM), a TPSA of 91.93 Å is indicative of a non-BBB permeant (requires a TPSA < 80 Å), however, as is the case with the other ligands prepared, gastrointestinal absorption is predicted to be high. All ligands are predicted to be P-glycoprotein substrates except for compound 19 and previously reported compound 5, suggesting that absorption from the gastrointestinal tract and across the BBB may be compromised as compounds may be effluxed, thus decreasing bioavailability. The physiochemical descriptors for the two most active ligands 20 and 28, compound 5 and donepezil 1 are summarised in Table 2. Ligand 20 (IC50 5.94 mM) which indicated 15-fold less activity than 28 is arguably more attractive as a leadlike compound with a TPSA and WLogP which places the ligand well within the range for BBB-permeants and with a molecular mass that is 45.07 g mol1 less than that of 28.

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Fig. 7. Schematic representation showing selected torsional angle of (a) donepezil 1, (b) 27 and (c) 20 during the 40 ns MD simulation at 310 K bound to AChE. Each colour represents a separate torsional angle for the ligands. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8. Boiled egg plot of WLogP vs. TPSA, yellow indicates BBB-permeant, white indicates gastrointestinal permeant, blue indicates P-glycoprotein þ, red indicates P-glycoprotein -.

Table 2 Physiochemical descriptors of selected ligands. Compound

MW (g/mol)

Num. rotatable bonds

H-bond acceptors

H-bond donors

TPSA (Å)

WLogP

5 20 28 Donepezil 1

409.47 404.50 449.57 379.49

7 6 7 6

6 3 5 4

0 1 1 0

65.07 59.27 91.93 38.77

2.73 2.61 3.86 3.83

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5. Conclusion A small library of fifteen potential AChE inhibitors was designed and prepared based upon the molecular skeleton of a potent lead compound 5 (IC50 0.03 mM) which we previously reported. Analogues were prepared by changing the ester linker present in 5 to that of an amide linker to reduce the metabolic liability associated with the ester functional group. Introduction of the amide linker appears to reduce activity, although it should be noted that the direct amide analogue 10 of compound 5 could not be accessed synthetically. That being said, two potential lead compounds were identified with AChE inhibitory IC50 activities below 10 mM, namely 20 (5.94 mM) and 28 (0.41 mM). The SAR and molecular modelling simulations suggest that although the use of the benzyl piperidine group affords distinctive binding to the CAS, concomitant binding to the PAS is critical for the development of an efficient inhibitor. This is evidenced as activities ranged from 0.41 mM to >100 mM (deemed not active), even though all compounds shared a common benzyl piperidine group with simple variations to the group at the PAS site. In the case of the most active compound 28, in silico assessments revealed that 28 and donepezil 1 share similar binding interactions. MD simulations, however, revealed that there are subtle differences in interactions, most notably, the indanone moiety in donepezil 1 is rigidly bound within the PAS site, whereas the tricyclic indenothiazole ring system of 28 appears to be able rotate by ~310 . The observed rotation arises as there is no clear interaction preference between the nitrogen and sulphur atoms of the thiazole ring. In contrast, compound 20 approximates the binding of donepezil 1 more closely, showing increased frequency of interactions with Tyr121, Phe288, Arg289, Phe330, Phe331 and Tyr334 and no major losses in frequency of interactions with other residues. In silico ADME predictions suggest that compound 20, although less active, is potentially more attractive as a lead compound with WLogP and TPSA values which indicative of being a potential BBB permeant. In addition, 20 has a molecular weight which is 45.07 g mol1 less than that of 28 and has less H-bond acceptors suggesting that it is more attractive as a lead compound. 6. Experimental 6.1. Chemistry 6.1.1. General methods All solvents, chemicals, and reagents were obtained commercially and used without further purification. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on Bruker AVANCE-III-300 instrument using CDCl3 and DMSO‑d6. CDCl3 contained tetramethylsilane as an internal standard. Chemical shifts, d, are reported in parts per million (ppm), and splitting patterns are given as singlet (s), doublet (d), triplet (t), quartet (q), or multiplet (m). Coupling constants, J, are expressed in hertz (Hz). Mass spectra were recorded in ESI mode on a Waters Synapt G2 Mass Spectrometer at 70 eV and 200 mA. Samples were prepared in acetonitrile (containing 0.1% formic acid) to an approximate concentration of 10 mg/mL. Infrared spectra were run on a Bruker ALPHA Platinum ATR spectrometer. The absorptions are reported on the wavenumber (cm1) scale, in the range 400e4000 cm1. Melting points were measured on a Stuart Melting Point SMP10 microscope and are uncorrected. The retention factor (Rf) values quoted are for thin layer chromatography (TLC) on aluminium-backed Macherey-Nagel ALUGRAM Sil G/UV254 plates pre-coated with 0.25 mm silica gel 60, spots were visualised with UV light and basic KMnO4 spray reagent. Chromatographic separations were performed on Macherey-Nagel Silica gel 60 (particle size 0.063e0.200 mm). Yields refer to isolated pure products unless stated otherwise.

6.1.2. Ethyl 1-benzylpiperidine-4-carboxylate 8 A mixture of ethyl isonipecotate 7 (10.00 g, 63.61 mmol, 1 eq.), benzyl chloride (7.69 mL, 66.79 mmol, 1.05 eq.) and triethylamine (26.45 mL, 190.8 mmol, 3 eq.) in anhydrous acetonitrile (100 mL) were refluxed for 3 h after which time the reaction mixture was left to cool to room temperature and the solvent was evaporated in vacuo. The obtained oil was diluted with ethyl acetate (100 mL) and washed with a 10% aqueous solution of sodium hydroxide (3  50 mL) and water (50 mL). The organic layer was dried (Na2SO4), filtered and evaporated in vacuo to afford ethyl 1benzylpiperidine-4-carboxylate 8 as an orange oil (88%) which was used as is without any further purification; Rf 0.42 (1:3 ethyl acetate: hexane); nmax (neat)/cm¡1 2944, 2801, 2760, 1728, 1448, 1168, 1046, 736, 698; 1H NMR (300 MHz, CDCl3); d 7.41e7.15 (m, 5H, ArH's), 4.13 (t, 2H, J ¼ 7.1 Hz, OCH2CH3), 3.51 (s, 2H, CH2Ph), 2.87 (dt, 2H, J ¼ 11.4, 3.1 Hz, CH2N), 2.30 (tt, 1H, J ¼ 10.9, 4.2 Hz, CHCO), 2.05 (td, 2H, J ¼ 11.3, 2.7 Hz, CH2N), 1.95e1.71 (m, 4H, CH2), 1.26 (t, J ¼ 7.1 Hz, OCH2CH3); 13C NMR (75 MHz, CDCl3); d 175.33, 138.49, 129.16, 128.28, 127.07, 63.35, 60.33, 53.02, 41.32, 28.39, 14.32; HRMS m/z (ESI) 248.1682 ([M þ H]þ requires 248.1645). 6.1.3. 1-Benzylpiperidine-4-carboxylic acid 9 To a stirring solution of sodium hydroxide (3.44 g, 86.02 mmol, 2 eq.) in methanol (100 mL) at room temperature was added ethyl 1benzylpiperidine-4-carboxylate 8 (10.01 g, 41.82 mmol, 1 eq.) in one portion. The resulting mixture was heated to reflux temperature and left to stir overnight. The resulting solution was cooled to room temperature and the solvent was evaporated in vacuo to afford a yellow solid. The solid was re-dissolved in distilled water (100 mL) and the pH adjusted to 5.5. The solvent was removed in vacuo to afford a yellow solid. A pre-mixed solution of ethanol: chloroform (1:1, 200 mL) was added and the mixture was left to stir for 1 h after which time the solids were collected by filtration and washed with chloroform (10 mL). The filtrate was collected, and the solvent was evaporated in vacuo to afford 1-benzylpiperidine-4carboxylic acid 9, as an off-white solid (98%) which was used as is without any further purification. (Yield 98%); Rf 0.07 (4:1 dichloromethane: methanol); mp 168  C; nmax (neat)/cm¡1 2969, 2864, 1602, 1448, 1382, 921, 756, 702, 659; 1H NMR (300 MHz, CDCl3); d 10.71 (s, 1H, OH), 7.38e7.27 (m, 5H, ArH's), 3.89 (s, 2H, CH2Ph), 3.14 (d, 2H, J ¼ 11.2 Hz, CH2N), 2.46 (t, 2H, J ¼ 8.9 Hz, CH2N), 2.34e2.21 (m, 1H, CHCO), 2.07e1.82 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 178.88, 132.25, 130.75, 128.74, 60.81, 51.50, 40.68, 26.72; HRMS m/z (ESI) 220.1353 ([M þ H]þ requires 220.1332). 6.1.4. (1-Benzylpiperidin-4-yl)(1H-imidazol-1-yl)methanone 6 1-Benzylpiperidine-4-carboxylic acid 9 (6.24 g, 28.48 mmol, 1 eq.) in dry dichloromethane (100 mL) was cooled to 0  C, and carbonyl diimidazole (5.57 g, 34.36 mmol, 1.2 eq.) was added portion-wise over 5 min. After 2 h, the mixture was diluted with dichloromethane (10 mL), and water (100 mL) was added carefully. The organic layer was washed with water (2  40 mL) and brine (30 mL), dried (Na2SO4) and evaporated in vacuo to afford (1benzylpiperidin-4-yl)(1H-imidazol-1-yl)methanone 6 as an offwhite solid (82%) which was used as is without any further purification; mp 119e121  C; nmax (neat)/cm¡1 2932, 1604, 1447, 1383, 1065, 922, 825, 753; 1H NMR (300 MHz, CDCl3); d 8.16 (s, 1H, imidazole NH), 7.46 (s, 1H, imidazole CH), 7.37e7.23 (m, 5H, ArH's), 7.09 (s, 1H, imidazole CH), 3.58 (s, 2H, CH2Ph), 3.00 (d, 2H, J ¼ 11.7 Hz, CH2N), 2.99e2.84 (m, 1H, CHCO), 2.27e2.09 (m, 2H, CH2N), 2.08e1.91 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 171.91, 136.18, 131.25, 129.28, 128.46, 127.46, 116.15, 77.16, 63.03, 52.38, 41.65, 28.54; HRMS m/z (ESI) 270.1707 ([M þ H]þ requires 270.1601).

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6.1.5. 2-Oximino-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one 13 To a solution of 5,6-dimethoxy-1-indanone 12 (1.50 g, 7.82 mmol, 1.0 eq.) in methanol (25 mL) at 40  C was added 96% isopentyl nitrite (1.31 mL, 9.38 mmol, 1.2 eq.) followed by concentrated aqueous hydrochloric acid (0.78 mL, 9.38 mmol, 1.2 eq.). The solution was stirred for 30 min during which time a precipitate formed. The precipitate was collected and dried to yield 2-oximino5,6-dimethoxy-2,3-dihydro-1H-inden-1-one 13 as a yellow solid (81%) which was used as is without any further purification; Rf 0.45 (3:1 ethyl acetate: hexane); mp 240  C (literature [18] 227e228  C); nmax (neat)/cm1 3189, 1694, 1579, 1498, 1449, 1304, 1233, 1119, 1029, 978, 915, 801, 746; 1H NMR (300 MHz, DMSO); d 12.41 (s, 1H, NOH), 7.17 (s, 1H, ArH), 7.16 (s, 1H, ArH), 3.89 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.64 (s, 2H, CH2); 13C NMR (75 MHz, DMSO); d 187.57, 155.98, 154.71, 149.32, 142.48, 130.54, 108.57, 104.51, 56.15, 56.13, 55.72, 55.69, 27.85; HRMS m/z (ESI) 292.1558 ([M þ H]þ requires 292.0761).

30 min at room temperature. The resulting mixture was refluxed for 12 h. The solution was then cooled down to room temperature and a 3 M ammonium hydroxide solution (64.55 mL, 129.10 mmol, 5 eq.) was added resulting in the precipitation of the product. The resulting mixture was left to stir for 30 min and the solids collected by filtration, followed by washing with distilled water (100 mL), after which time the solid was dried in vacuo, affording 5,6dimethoxy-8H-indeno[1,2-d]thiazol-2-amine 29 as a yellow solid (83%) which was used as is without further purification; Rf 0.27 (3:1 ethyl acetate: hexane); mp 204  C (decomp.); nmax (neat)/cm¡1 3376, 3111, 2933, 1524, 1456, 1377, 1271, 1203, 1148, 1097, 750; 1H NMR (300 MHz, DMSO‑‑d6); d 7.16 (s, 1H, ArH), 7.06 (s, 2H, NH2), 6.98 (s, 1H, ArH), 3.79 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.58 (s, 2H, CH2); 13C NMR (75 MHz, DMSO‑‑d6); d 172.75, 156.01, 148.05, 146.37, 137.51, 130.80, 120.70, 110.11, 102.13, 55.96, 55.67, 31.98; HRMS m/z (ESI) 249.0697 ([M þ H]þ requires 249.0692).

6.1.6. 2-Amino-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one hydrochloride 11 A suspension of 2-oximino-5,6-dimethoxy-2,3-dihydro-1Hinden-1-one 13 (0.21 g, 0.93 mmol, 1.0 eq.), concentrated aqueous hydrochloric acid (0.12 mL, 1.40 mmol, 1.5 eq.) and 10% palladium on carbon (0.05 g, 50 mg mmol1) in ethanol (15 mL) was hydrogenated at atmospheric pressure and room temperature until the incorporation of the hydrogen was complete. The catalyst was then removed by filtration and washed with ethanol (3  25 mL). The solvent of the filtrate was then removed in vacuo to afford 2-amino5,6-dimethoxy-2,3-dihydro-1H-inden-1-one hydrochloride 11 as a light yellow powder (95%) which was used as is without any further purification; Rf 0.54 (3:1 ethyl acetate: hexane); mp 240  C decomposed (literature [19] 240  C decomposed); nmax (neat)/ cm1 2915, 1708, 1590, 1496, 1318, 1270, 1128, 1087, 1014, 801; 1H NMR (300 MHz, DMSO); d 8.90 (s, 3H, NHþ 3 ), 7.20 (s, 1H, ArH), 7.14 (s, 1H, ArH), 4.17 (s, 1H, COCH), 3.88 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.46 (d, 1H, J ¼ 7.9 Hz, CH2aCHNHþ 3 ), 3.07 (dd, 1H, J ¼ 16.8, 13 4.6 Hz, CH2bCHNHþ C NMR (75 MHz, DMSO); d 198.45, 156.30, 3 ); 149.66, 147.21, 126.38, 108.40, 104.30, 56.24, 56.21, 55.84, 55.81, 53.32, 31.04; HRMS m/z (ESI) 208.1000 ([M þ H]þ requires 208.0968).

6.1.9. General method for amide couplings A mixture of amine (1.00 mmol, 1 eq.), (1-benzylpiperidin-4yl)(1H-imidazol-1-yl) methanone 6 (1.11 mmol, 1.1 eq.), 4dimethylaminopyridine (0.60 mmol, 0.6 eq.) and triethylamine (1.00 mmol, 1 eq.) in anhydrous dichloromethane (15 mL) was left to stir overnight at room temperature. The mixture was then refluxed for 2 h after which time it was left to cool down to room temperature. A solution of 3 M aqueous sodium hydroxide (50 mL) was added to the reaction mixture. The organic layer was separated and the aqueous layer was washed with dichloromethane (2  30 mL). The organic layers were combined and washed with distilled water (30 mL). The organic layer was dried (Na2SO4), filtered and evaporated in vacuo. The product was purified by column chromatography.

6.1.7. 2-Bromo-5,6-dimethoxy-1-indanone 30 To a stirring solution of 5,6-dimethoxy-1-indanone 12 (10.00 g, 52.03 mmol, 1 eq.) in methanol (100 mL), was added bromine (2.82 mL, 54.63 mmol, 1.05 eq.) drop-wise. After addition of bromine the product precipitated out of solution. The mixture was left to stir for 30 min after which time the precipitate was filtered off, washed with ice cold methanol (50 mL), and dried in vacuo to afford 2-bromo-5,6-dimethoxy-1-indanone 30 as a light-yellow powder (84%) which was used as is without any further purification; Rf 0.72 (3:1 ethyl acetate: hexane); mp 162e163  C [15]; nmax (neat)/cm¡1 2959, 2688, 1687, 1583, 1497, 1309, 1261, 1218, 1107, 1015, 727; 1H NMR (300 MHz, CDCl3); d 7.19 (s, 1H, ArH), 6.82 (s, 1H, ArH), 4.61 (dd, 1H, J ¼ 2.9 & 7.3 Hz, CHBr), 3.95 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.72 (dd, 1H, J ¼ 7.2 & 17.9 Hz, CH2aCHBr), 3.30 (dd, 1H, J ¼ 2.98 & 17.9 Hz, CH2bCHBr); 13C NMR (75 MHz, CDCl3); d 198.17, 156.75, 150.19, 146.73, 126.44, 107.27, 105.24, 56.49, 56.46, 56.28, 56.25, 44.70, 37.86. 6.1.8. 5,6-Dimethoxy-8H-indeno[1,2-d]thiazol-2-amine 29 To a suspension of thiourea (1.97 g, 25.89 mmol, 1 eq.) in distilled water (100 mL) was added 2-bromo-5,6-dimethoxy-1indanone 30 (7.00 g, 25.82 mmol, 1 eq.) in three portions over

6.1.9.1. N-[2-(1H-indole-3-yl)ethyl]-1-benzylpiperidine-4carboxamide 14. (Yield, 91%); Off-white solid; Rf 0.42 (9:1 dichloromethane: methanol); mp 144e145  C; nmax (neat)/cm¡1 3309, 2929, 2816, 1630, 1532, 1445, 1348, 1294, 1207, 1105, 1031, 990, 922, 741, 702; 1H NMR (300 MHz, CDCl3); d 8.45 (s, 1H, indole NH), 8.45 (s, 1H, NHCO), 7.59 (d, 1H, J ¼ 7.8 Hz, ArH's), 7.39e7.24 (m, 6H, ArH's), 7.20 (t, 1H, J ¼ 7.4 Hz, ArH), 7.11 (t, 1H, J ¼ 7.2 Hz, ArH), 6.98 (d, 1H, J ¼ 1.9 Hz, ArH), 5.66 (t, 1H, J ¼ 4.5 Hz), 3.58 (q, 2H, J ¼ 6.5 Hz, CH2NHCO), 3.51 (s, 2H, CH2Ph), 3.01e2.86 (m, 4H, CH2), 2.08e1.92 (m, 3H, CHCO & CH2), 1.82e1.62 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 175.02, 136.54, 129.38, 128.40, 127.42, 127.37, 122.26, 122.24, 119.49, 118.79, 112.91, 111.47, 63.08, 52.94, 43.06, 39.64, 28.71, 25.38; HRMS m/z (ESI) 362.2209 ([M þ H]þ requires 362.2227).

6.1.9.2. 1-Benzyl-N-[2-(naphthalen-1-ylamino)ethyl] piperidine-4carboxamide 15. (Yield, 90%); Green solid; Rf 0.61 (9:1 dichloromethane: methanol); mp 120e121  C; nmax (neat)/cm¡1 3402, 3314, 2929, 1628, 1582, 1538, 1412, 1292, 1135, 954, 753, 689; 1H NMR (300 MHz, CDCl3); d 7.89e7.82 (m, 1H, ArH), 7.81e7.74 (m, 1H, ArH), 7.47e7.40 (m, 2H, ArH's), 7.36e7.24 (m, 6H, ArH's), 7.22 (d, J ¼ 8.2 Hz, 1H, ArH), 6.51 (d, J ¼ 7.4 Hz, 1H, ArH), 6.15 (s, 1H, NHCO), 5.20 (s, 1H, NH), 3.64 (q, J ¼ 5.8 Hz, 2H,CH2NHCO), 3.47 (s, 2H, CH2Ph), 3.36 (t, J ¼ 5.4 Hz, 2H, CH2NH), 2.89 (d, J ¼ 11.5 Hz, 2H, CH2N), 2.17e1.89 (m, 3H, CHCO & CH2N), 1.84e1.72 (m, 4H, CH2); 13 C NMR (75 MHz, CDCl3); d 176.78, 143.66, 134.39, 129.30, 128.59, 128.37, 127.33, 126.67, 125.92, 124.94, 123.36, 120.46, 117.19, 103.46, 63.02, 52.84, 45.39, 42.93, 42.89, 38.87, 28.72; HRMS m/z (ESI) 388.2424 ([M þ H]þ requires 388.2383).

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6.1.9.3. 1-Benzyl-N-(2-morpholinoethyl) piperidine-4-carboxamide 16. (Yield, 85%); Yellow solid; Rf 0.30 (9:1 dichloromethane: methanol); mp 89e90  C; nmax (neat)/cm¡1 3266, 2928, 1638, 1540, 1441, 1227, 1114, 1002, 913, 863, 732, 698; 1H NMR (300 MHz, CDCl3); d 7.35e7.19 (m, 5H, ArH's), 6.15 (s, 1H, NHCO), 3.68 (m, 4H, CH2O), 3.52 (s, 2H, CH2Ph), 3.32 (dd, 2H, J ¼ 11.2, 5.7 Hz, CH2NHCO), 2.94 (d, 2H, J ¼ 11.6 Hz, CH2N), 2.49e2.38 (m, 6H, (CH2)2NCH2, & CH2N), 2.18e1.98 (m, 3H, CHCO & CH2N), 1.89e1.67 (m, 4H, CH2); 13 C NMR (75 MHz, CDCl3); d 175.05, 137.65, 129.33, 128.34, 127.29, 66.98, 63.14, 57.07, 53.37, 52.99, 43.01, 35.51, 28.78; HRMS m/z (ESI) 332.2341 ([M þ H]þ requires 332.2332).

6.1.9.8. 1-Benzyl-N-(pyridine-2-ylmethyl) piperidine-4-carboxamide 21. (Yield, 77%); Yellow viscous oil; Rf 0.33 (9:1 dichloromethane: methanol); nmax (neat)/cm¡1 3343, 2940, 2792, 1639, 1592, 1537, 1429, 1295, 1214, 1133, 993, 732; 1H NMR (300 MHz, CDCl3); d 8.50 (d, 1H, J ¼ 4.7 Hz, ArH), 7.63 (td, 1H, J ¼ 7.7, 1.6 Hz, ArH), 7.36e7.13 (m, 7H, ArH's), 6.97 (s, 1H, NHCO), 4.53 (d, 2H, J ¼ 4.8 Hz, CH2NHCO), 3.53 (s, 2H, CH2Ph), 2.96 (d, 2H, J ¼ 11.6 Hz, CH2N), 2.30e2.16 (m, 1H, CHCO), 2.07 (m, 2H, CH2N), 1.96e1.74 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 175.11, 156.45, 149.02, 137.68, 136.86, 129.34, 128.35, 127.29, 122.43, 122.14, 63.11, 53.00, 44.31, 42.99, 28.74; HRMS m/z (ESI) 310.1939 ([M þ H]þ requires 310.1914).

6.1.9.4. 1-Benzyl-N-(2-methoxybenzyl) piperidine-4-carboxamide 17. (Yield, 99%); Yellow crystalline solid; Rf 0.50 (9:1 dichloromethane: methanol); mp 120  C; nmax (neat)/cm¡1 3274, 2932, 1642, 1546, 1489, 1445, 1236, 1114, 1025, 985, 736, 696; 1H NMR (300 MHz, CDCl3); d 7.34e7.20 (m, 7H, ArH's), 6.89 (dd, 2H, J ¼ 15.1, 7.7 Hz, ArH's), 6.06 (s, 1H, NHCO), 4.43 (d, 2H, J ¼ 5.7 Hz, CH2NHCO), 3.84 (s, 3H, CH3O), 3.50 (s, 2H, CH2Ph), 2.93 (d, 2H, J ¼ 11.7 Hz, CH2N), 2.18e1.93 (m, 3H, CHCO & CH2N), 1.90e1.67 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 174.61, 157.61, 138.04, 129.78, 129.25, 128.90, 128.31, 127.18, 126.40, 120.78, 110.38, 63.19, 55.44, 55.38, 53.10, 43.27, 39.35, 28.86; HRMS m/z (ESI) 339.2069 ([M þ H]þ requires 339.2067).

6.1.9.9. 1-Benzyl-N-(pyridine-3-ylmethyl) piperidine-4-carboxamide 22. (Yield, 59%); Yellow viscous oil; Rf 0.23 (9:1 dichloromethane: methanol); nmax (neat)/cm¡1 3312, 3029, 2947, 2921, 2793, 2750, 1635, 1533, 1430, 1296, 1218, 1132, 1020, 978, 704, 657; 1 H NMR (300 MHz, CDCl3); d 8.45 (d, 1H, J ¼ 4.7 Hz, ArH), 8.45 (s, 1H, ArH), 7.57 (d, 1H, J ¼ 7.8 Hz, ArH), 7.32e7.18 (m, 6H, ArH's), 6.45 (s, 1H, NHCO), 4.40 (d, 2H, J ¼ 5.9 Hz, CH2NHCO), 3.49 (s, 2H, CH2Ph), 2.92 (d, 2H, J ¼ 11.7 Hz, CH2N), 2.22e2.09 (m, 1H, CHCO), 2.00 (td, 2H, J ¼ 11.1, 3.5 Hz, CH2N), 1.87e1.69 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 175.30, 149.05, 148.80, 137.87, 135.59, 134.35, 129.21, 128.33, 127.24, 123.68, 63.10, 52.96, 43.05, 40.83, 28.83; HRMS m/z (ESI) 310.1939 ([M þ H]þ requires 310.1914).

6.1.9.5. 1-Benzyl-N-(thiazol-2-yl) piperidine-4-carboxamide 18. (Yield, 59%); Off-white solid; Rf 0.50 (9:1 dichloromethane: methanol); mp 179e181  C; nmax (neat)/cm¡1 3156, 2928, 1679, 1560, 1494, 1442, 1368, 1329, 1290, 1267, 1186, 1167, 1141, 1108, 1075, 1010, 964, 799, 743, 726, 700; 1H NMR (300 MHz, CDCl3); d 12.53 (s, 1H, NHCO), 7.50 (d, 1H, J ¼ 3.6 Hz, CHN), 7.38e7.23 (m, 5H, ArH's), 7.02 (d, 1H, J ¼ 3.6 Hz, CHS), 3.55 (s, 2H, CH2Ph), 3.01 (d, 2H, J ¼ 11.0 Hz, CH2N), 2.59e2.46 (m, 1H, CHCO), 2.16e1.84 (m, 6H, CH2N & CH2); 13C NMR (75 MHz, CDCl3); d 173.68, 160.50, 138.30, 136.24, 129.15, 128.38, 127.23, 113.74, 63.27, 53.08, 43.09, 28.54; HRMS m/z (ESI) 302.1362 ([M þ H]þ requires 302.1322).

6 .1. 9 .10 . 1 - B e n z yl - N - ( p yr i d i n e - 4 - yl m e t hyl ) p ip er i d i n e - 4 carboxamide 23. (Yield, 77%); Yellow viscous oil; Rf 0.33 (9:1 dichloromethane: methanol); nmax (neat)/cm¡1 3290, 2917, 2800, 1638, 1542, 1428, 1374, 1302, 1073, 990, 795, 736, 693, 638; 1H NMR (300 MHz, CDCl3); d 8.48 (d, 2H, J ¼ 4.8 Hz, pyridine CH's), 7.35e7.20 (m, 5H, ArH's), 7.12 (d, 2H, J ¼ 5.0 Hz, pyridine CH's), 6.48 (s, 1H, NHCO), 4.41 (d, 2H, J ¼ 5.9 Hz, CH2NHCO), 3.52 (s, 2H, CH2Ph), 2.95 (d, 2H, J ¼ 11.2 Hz, CH2N), 2.21 (ddd, 1H, J ¼ 15.2, 10.1, 4.8 Hz, CHCO), 2.04 (td, 2H, J ¼ 10.8, 2.6 Hz, CH2N), 1.90e1.73 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 175.40, 150.00, 147.82, 137.70, 129.26, 128.37, 127.32, 122.26, 63.07, 52.91, 42.99, 42.15, 28.83; HRMS m/z (ESI) 310.1939 ([M þ H]þ requires 310.1914).

6.1.9.6. 1-Benzyl-N-(3,4,5-trimethoxyphenethyl)piperidine-4carboxamide 19. (Yield, 93%); Light yellow solid; Rf 0.56 (9:1 dichloromethane: methanol); mp 119e121  C; nmax (neat)/cm¡1 3327, 2926, 1635, 1589, 1547, 1509, 1454, 1435, 1324, 1245, 1128, 999, 817, 714, 676; 1H NMR (300 MHz, CDCl3); d 7.32e7.20 (m, 5H, ArH's), 6.38 (s, 2H, ArH's), 5.69 (s, 1H, NHCO), 3.82 (s, 6H, CH3O), 3.81 (s, 3H, CH3O), 3.53e3.44 (m, 4H, CH2Ph & CH2NHCO), 2.91 (d, 2H, J ¼ 11.6 Hz, CH2N), 2.73 (t, 2H, J ¼ 7.0 Hz, PhCH2CH2NH), 2.15e1.91 (m, 3H, CHCO & CH2N), 1.85e1.63 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 175.04, 153.34, 136.58, 134.69, 129.23, 128.33, 127.26, 105.66, 105.64, 63.09, 60.88, 56.18, 52.93, 43.07, 40.47, 36.13, 28.83; HRMS m/z (ESI) 413.2458 ([M þ H]þ requires 413.2453). 6.1.9.7. 1-Benzyl-N-(1-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) piperidine-4-carboxamide 20. (Yield, 34%); Off-white crystalline solid; Rf 0.33 (9:1 dichloromethane: methanol); mp 235e236  C; nmax (neat)/cm¡1 3228, 3185, 2925, 1684, 1640, 1612, 1587, 1451, 1296, 1195, 1109, 957, 746, 692; 1H NMR (300 MHz, CDCl3); d 8.33 (s, 1H, NHCO), 7.49e7.19 (m, 10H, ArH's), 3.51 (s, 2H, CH2Ph), 3.04 (d, 3H, J ¼ 0.8 Hz, NCH3), 2.90 (d, 2H, J ¼ 11.0 Hz, CH2N), 2.33 (dt, 1H, J ¼ 14.4, 7.3 Hz, CHCO), 2.19 (s, 3H, CCH3), 2.09e1.92 (m, 2H, CH2N), 1.90e1.77 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 174.65, 161.93, 149.69, 134.70, 129.37, 128.34, 127.22, 127.06, 124.34, 109.04, 63.05, 52.84, 42.56, 36.32, 28.77, 12.66; HRMS m/z (ESI) 405.2292 ([M þ H]þ requires 405.2285).

6.1.9.11. 1-Benzyl-N-(furan-2-ylmethyl) piperidine-4-carboxamide 24. (Yield, 97%); Off-white crystalline solid; Rf 0.46 (9:1 dichloromethane: methanol); mp 110  C; nmax (neat)/cm¡1 3308, 2939, 2922, 2815, 2767, 1639, 1535, 1450, 1256, 1143, 1109, 1076, 1011, 981, 911, 822, 749, 697, 643; 1H NMR (300 MHz, CDCl3); d 7.36e7.20 (m, 6H, ArH's), 6.30 (dd, 1H, J ¼ 3.1, 1.9 Hz, CHCHCH), 6.19 (d, 1H, J ¼ 2.9 Hz, CHCHC), 6.01 (s, 1H, NHCO), 4.41 (d, 2H, J ¼ 5.4 Hz, CH2), 3.51 (s, 2H, CH2Ph), 2.94 (d, 2H, J ¼ 11.7 Hz, CH2N), 2.20e1.95 (m, 3H, CHCO & CH2N), 1.89e1.68 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 174.83, 151.43, 142.23, 137.87, 129.26, 128.34, 127.25, 110.53, 107.47, 63.12, 52.97, 43.03, 36.49, 28.75; HRMS m/z (ESI) 299.1740 ([M þ H]þ requires 299.1754). 6.1.9.12. 1-Benzyl-N-(1,2,3,4-tetrahydro-naphthalen-1-yl) piperidine-4-carboxamide 25. (Yield, 53%); Pink crystalline solid; Rf 0.56 (9:1 dichloromethane: methanol); mp 135e136  C; nmax (neat)/ cm¡1 3256, 2929, 1637, 1539, 1446, 1223, 1119, 963, 737, 695; 1H NMR (300 MHz, CDCl3); d 7.37e7.04 (m, 9H, ArH's), 5.80 (d, 1H, J ¼ 8.3 Hz, NHCO), 5.22e5.12 (m, CHN), 3.52 (s, 2H, CH2Ph), 2.95 (d, 2H, J ¼ 11.6 Hz, CH2N), 2.77 (q, 2H, J ¼ 5.7 Hz, PhCH2CH2), 2.19e1.96 (m, 3H, CHCO & CH2N), 1.92e1.71 (m, 8H, CH2); 13C NMR (75 MHz, CDCl3); d 174.25, 137.66, 136.86, 129.27, 128.61, 128.35, 127.33, 127.24, 126.36, 63.15, 53.03, 47.19, 43.30, 30.29, 29.33, 28.91, 20.09; HRMS m/z (ESI) 349.2260 ([M þ H]þ requires 349.2274).

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6.1.9.13. N-[2-(1H-imidazol-4-yl)ethyl]-1-benzylpiperidine-4carboxamide 26. (Yield, 38%); Yellow viscous oil; Rf 0.13 (9:1 dichloromethane: methanol); nmax (neat)/cm¡1 3246, 3084, 2938, 2796, 2764, 1632, 1555, 1442, 1336, 1226, 1104, 987, 931, 801, 728, 698, 659; 1H NMR (300 MHz, CDCl3); d 9.74 (s, 1H, imidazole NH), 7.56 (s, 1H, imidazole CH), 7.32e7.18 (m, 5H, ArH's), 6.95 (t, 1H, J ¼ 5.4 Hz, NHCO), 6.77 (s, 1H, imidazole CH), 3.53e3.44 (m, 4H, CH2Ph & CH2NHCO), 2.90 (d, 2H, J ¼ 11.4 Hz, CH2N), 2.78 (t, 2H, J ¼ 6.5 Hz, CH2CH2NH), 2.17e2.04 (m, 1H, CHCO), 1.97 (td, 2H, J ¼ 11.1, 3.6 Hz, CH2N), 1.81e1.64 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); d 175.71, 137.95, 135.76, 134.94, 129.29, 128.34, 127.24, 116.35, 63.22, 53.09, 43.24, 39.36, 28.86, 26.80; HRMS m/z (ESI) 313.2023 ([M þ H]þ requires 313.2023). 6.1.9.14. 1-Benzyl-N-(2-(piperidin-1-yl)ethyl)piperidine-4carboxamide 27. (Yield, 59%); Yellow viscous oil; Rf 0.24 (9:1 dichloromethane: methanol); nmax (neat)/cm¡1 3294, 2933, 2770, 1637, 1551, 1443, 1300, 1126, 1038, 785, 726, 687; 1H NMR (300 MHz, CDCl3); d 7.33e7.21 (m, 5H, ArH's), 6.46 (s, 1H, NHCO), 3.50 (s, 2H, CH2Ph), 3.35 (dd, 2H, J ¼ 11.2, 5.6 Hz, CH2NHCO), 2.93 (d, 2H, J ¼ 11.6 Hz, CH2N), 2.48 (dd, 6H, J ¼ 13.3, 7.3 Hz, (CH2)2NCH2), 2.12 (m, 1H, CHCO), 2.00 (td, 2H, J ¼ 11.4, 2.8 Hz, CH2N), 1.88e1.70 (m, 4H, CH2), 1.61 (dt, 4H, J ¼ 10.7, 5.5 Hz, CH2), 1.51e1.41 (m, 2H, CH2); 13C NMR (75 MHz, CDCl3); d 175.25, 138.41, 129.21, 128.26, 127.06, 63.32, 57.16, 54.28, 53.25, 43.36, 35.68, 29.00, 25.69, 24.17; HRMS m/z (ESI) 330.2541 ([M þ H]þ requires 330.2540). 6.1.9.15. 1-Benzyl-N-(5,6-dimethoxy-8H-indeno[1,2-d]thiazol-2-yl) piperidine-4-carboxamide 28. A mixture of 5,6-dimethoxy-8Hindeno[1,2-d]thiazole-2-amine 11 (0.25 g, 1.00 mmol, 1 eq.), (1benzylpiperidin-4-yl)(1H-imidazol-1-yl) methanone 9 (0.30 g, 1.11 mmol, 1.1 eq.) and sodium hydride (0.08 g, 2.03 mmol, 2 eq., 60% in oil) in anhydrous tetrahydrofuran (15 mL) was refluxed overnight after which time the reaction mixture was left to cool down to room temperature. The reaction was quenched with the slow addition of a saturated aqueous solution of sodium sulfate (1 mL). A solution of 3 M aqueous sodium hydroxide was added and left to stir for 5 min. The organic layer was separated and the aqueous layer was washed with dichloromethane (2  30 mL). The organic layers were combined, washed with distilled water (30 mL), dried (Na2SO4), filtered and evaporated in vacuo. The product was purified by column chromatography. (Yield, 12%); Brown solid; Rf 0.55 (9:1 dichloromethane: methanol); mp 218  C (decomp.); nmax (neat)/cm¡1 2932, 1687, 1536, 1452, 1383, 1272, 1208, 1142, 1070, 989, 846, 738, 697; 1H NMR (300 MHz, CDCl3); d 10.29 (s, 1H, NHCO), 7.33e7.19 (m, 5H, ArH's), 7.20 (s, 1H, ArH), 7.13 (s, 1H, ArH), 3.95 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.74 (s, 2H, indene CH2), 3.35 (s, 2H, CH2Ph), 2.79 (d, 2H, J ¼ 11.3 Hz, CH2N), 2.34e2.20 (m, 1H, CHCO), 1.89e1.56 (m, 6H, CH2N & CH2); 13C NMR (75 MHz, CDCl3); d 172.89, 162.30, 155.29, 148.84, 147.72, 138.28, 137.98, 129.90, 129.18, 129.08, 128.35, 127.23, 109.48, 102.35, 63.11, 56.45, 56.37, 52.47, 42.88, 32.50, 28.52; HRMS m/z (ESI) 450.1859 ([M þ H]þ requires 450.1846). 6.2. Bioactivity screening Prior to screening, all compounds were dissolved in dimethyl sulfoxide (DMSO) due to the compounds being insoluble in water to a stock concentration of 10 mM. The final concentration of DMSO in the reaction was <1%. 6.2.1. Cholinesterase inhibitory activity Cholinesterase inhibitory activity for EeAChE and eqBuChE were determined using the 5,5-dithiobis-2-nitrobenzoic acid (DTNB) assay as described by Ellman [22] and modified by Eldeen and co-

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workers [23]. Three buffers were prepared: Buffer A e 50 mM Trishydrochloride (pH 8); Buffer B - 50 mM Tris-hydrochloride (pH 8), containing 0.1% bovine serum albumin; Buffer C - 50 mM Trishydrochloride (pH 8), fortified with 0.1 M sodium chloride and 0.02 M magnesium chloride. Into 96-well plates were pipetted: 25 mL acetylthiocholine iodide (15 mM in distilled water), 125 mL DTNB (3 mM in buffer C), 50 mL buffer B and either 25 mL buffer A (negative control), galantamine (positive control at 10 mM) or compounds 1, 14e28 (0.1e1 mM). Absorbance was measured at 405 nm (four times) to account for baseline noise. An aliquot of 25 mL EeAChE (0.2 U/mL in buffer A) was pipetted into the plates and the absorbance measured every 45 s for fifteen cycles. The EeAChE inhibition (% relative to negative control) was determined through the rate of the reaction (correcting for spontaneous colour changes) relative to the negative control. In the case of the BuChE inhibitory activity assay, EeAChE was replaced with eqBuChE (0.2 U/ mL in buffer A) as well as acetylcholine iodide with butyrylcholine iodide (15 mM in distilled water). 6.2.2. Cytotoxicity screening Cytotoxicity was assessed using the SRB staining assay on the SH-SY5Y neuroblastoma cell line as described by Vichai and Kirtikara with minor modifications [24]. Although the SH-SY5Y cell line is cancerous in nature, it does present as a model of a neurological cellular environment. The SH-SY5Y cell line was cultured in DMEM/ Ham's F12 nutrient medium (1:1) supplemented with 10% foetal calf serum (FCS) in 75 mL flasks at 37  C and 5% CO2 in a humidified incubator. Culture flasks with confluent cells were rinsed with phosphate buffered saline and harvested using TrypLe™Express to detach the cells. Detached cells were centrifuged (200g, 5 min), counted using the trypan blue exclusion assay (0.1%), and diluted to 1  105 cells/mL in 10% FCS-fortified medium. The cell suspension (100 mL) was seeded into sterile, clear 96-well plates, and incubated overnight to allow the cells to attach. Blank wells contained 200 mL FCS (5%)-fortified media without cells to account for background noise and sterility. Attached cells were exposed to 100 mL medium (negative control), compounds 1, 14e28 (0.01e100 mM) or saponin (1%; positive control) prepared in FCS negative medium for 72 h at 37  C and 5% CO2 in a humidified incubator. Cells were fixed in the wells by adding 50 mL trichloroacetic acid (50%) and left overnight at 4  C. Plates containing the fixed cells were washed three times with tap water and stained using 100 mL SRB solution (0.057% in 1% acetic acid) for 30 min at room temperature in the dark. Stained cells were washed four times with 150 mL acetic acid (1%) and airdried. The bound dye was eluted using 200 mL Tris-buffer (10 mM, pH 10.5) and the absorbance measured at 510 nm (reference 630 nm) using a ELx 800 microplate plate reader (Bio-Tek Instruments, Inc.). The blank value was subtracted from all the other values and the cell density was expressed relative to the negative control as a percentage. 6.2.3. Statistics Assays were performed with three intra- and inter-replicates. Statistical analyses were performed using Graph-Pad Prism 5.0 (GraphPad). The IC50 values were determined using non-linear regression analysis (variable slope). 6.3. Molecular modelling Ligand structures were prepared using ligprep, with protonation states being predicted using epic with a target pH of 7.0 ± 2.0. The protein structure was prepared using protein preparation wizard €dinger [30,31] in which protonation states were from Schro assigned followed by an energy minimisation to relieve unfavourable constraints. Molecular docking was done using Glide extra

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precision (XP) scoring function and ligand structures were treated as flexible, which allowed for sampling of ring conformations and nitrogen inversion, to obtain 5 poses for each ligand. The free binding energy was calculated by submitting each docked pose complex to the Prime MM-GBSA tool with the VSGB solvation model and OPLS3 force field [26,27,32]. The complexes with the highest binding pose score for donepezil 1, 20 and 28 were submitted for MD simulations using Des€dinger Suite and the OPLS3 force field mond found within the Schro [33e35]. Solvent molecules within the binding pocket from the docking procedure were kept and the system was further solvated using the Simple point-charge (SPC) water model for a simulation box with boundaries 10 Å away from the protein, with an orthorhombic box containing approximately 52,000 atoms. The minimisation of the system was done using steepest descent, followed by default relaxation protocol (see SI) and then MD simulation was done for 40 ns with NPT conditions using the Berendsen thermostat (310 K, 1.103 bar) and particle mesh Ewald (PME) electrostatics with a cut-off of 9 Å. Frames were extracted every 200 ps. Analysis of the MD simulations was done using the simulation interaction diagram €dinger Suite [36]. Using Prime MM-GBSA the tool within the Schro average binding free energy was calculated for each frame of each simulation e see SI. Acknowledgements This work was supported by the National Research Foundation (NRF) of South Africa (Thuthuka grant number 106959), the University of Pretoria (Research and Development Program) and the Council for Scientific and Industrial Research (CSIR), South Africa. Opinions expressed in this publication and the conclusions arrived at, are those of the authors, and are not necessarily attributed to the NRF. The authors would like to gratefully acknowledge Ms Madelien Wooding (University of Pretoria) for LC-MS services; and the Centre for High Performance Computing (CHPC) for access to the €dinger Suite and computational resources used in this work. Schro

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